Nonsymbiotic plant hemoglobins to maintain cell energy status

ABSTRACT

Nonsymbiotic hemoglobins are broadly present across evolution; however, the function of these proteins is unknown. Cultured maize cells have been transformed to constitutively express a barley hemoglobin gene in either the sense (HB + ) or antisense (HB − ) orientation. Hemoglobin protein in the transformed cell lines was correspondingly higher or lower than in wild type cells under normal atmospheric conditions. Limiting oxygen availability, by placing the cells in a nitrogen atmosphere for 12 hours, had little effect on the energy status of cells constitutively expressing hemoglobin, but had a pronounced effect on both wild type and HB −  cells, where ATP levels declined by 27% and 61% respectively. Energy charge was relatively unaffected by the treatment in HB +  and wild type cells, but was reduced from 0.91 to 0.73 in HB −  cells suggesting that the latter were incapable of maintaining their energy status under the low oxygen regime. Similar results were observed with  P. aeruginosa  cells transformed with an Hb expression vector. It is suggested that nonsymbiotic hemoglobins act to maintain the energy status of cells in low oxygen environments and that they accomplish this effect by promoting glycolytic flux through NADH oxidation, resulting in increased substrate level phosphorylation. Nonsymbiotic hemoglobins are likely ancestors of an early form of hemoglobin that sequestered oxygen in low oxygen environments, providing a source of oxygen to oxidize NADH to provide ATP for cell growth and development. This in turn suggests that cells containing increased levels of Hb protein will survive longer under low oxygen tension or high energy demand.

This application is a National Phase entry of PCT CA99/00587, having aninternational filing date of Jun. 24, 1999 and this application claimspriority under 35 USC §119(e) to U.S. Ser. No. 60/106,638, filed Nov. 2,1998 and to U.S. Ser. No. 60/090,929, filed Jun. 26, 1998.

The present invention relates generally to the field of expressionvectors and transgenic organisms.

BACKGROUND OF THE INVENTION

Hemoglobins are widespread throughout the biosphere (Wittenberg andWittenberg, 1990. Annu Rev Biophys Biophys Chem 19:217-241). They arefound in a broad range of organisms from bacteria, through unicellulareukaryotes, to plants and animals, suggesting that they predatedivergence of life into plant and animal forms. Plant hemoglobins havebeen classified into symbiotic and nonsymbiotic types (Appleby. 1992,Sci Progress 70:365-398): symbiotic hemoglobins are found in plants thatare capable of participating in microbial symbioses, where they functionin regulating oxygen supply to nitrogen fixing bacteria; nonsymbiotichemoglobins have only recently been discovered and are thought to be theevolutionary predecessors of the more specialized symbioticleghemoglobins. The ubiquitous nature of nonsymbiotic hemoglobins isevidenced by their broad presence across the plant kingdom (Appleby,1985, Nitrogen Fixation and CO ₂ Metabolism, eds. Ludden and Bums, pp.41-51) and the widespread presence and long evolutionary history ofplant hemoglobins suggest a major role for them in the life of plants.

Specifically, plant hemoglobins have been known to exist in the rootnodules of legumes for almost 60 years (Kubo, 1939, Acta Phitochem11:19-200; Keilen and Wang, 1945, Nature 155:227-229). Over the years,hemoglobins have been positively identified in three non-leguminousdicotyledonous plants; Parasponia andersonil, Tream tomentosa, andCasuarina glauca (Appleby et al., 1983, Science 220:951-954; Bogusz etal., 1988, Nature 331:178-180; Kort et al., 1998, FEBS Lett 180:55-60).Recently, an Hb cDNA from badey was isolated and the gene wasdemonstrated to be expressed in seed and root issues under anaerobicconditions (Taylor et al., 1994. Plant Mol Biol 24:853-862), providingfurther evidence to support the contention that plant hemoglobins have acommon origin (Landsmann et al., 1986. Nature 324:166-168). Since Hb hasnow been demonstrated to occur in two of the major divisions of theplant kingdom, it is likely that an Hb gene is present in the genome ofall higher plants (Brown et al., 1984, J Mol Evol 21:19-32; Bogusz etal., 1988; Appleby, 1992, Sci Progress 76:365-398; Taylor et al., 1994;Andersson et al., 1996, Proc Natl Acad Sci USA 93:427-431; Hardison,1996, Proc Natl Acad Sci USA 93:5675-5682).

Very little, however, is known about the function of Hb, although it hasbeen proposed that nonsymbiotic hemoglobins may act either as oxygencarriers to facilitate oxygen diffusion, or oxygen sensors to regulateexpression of anaerobic proteins during periods of low oxygen supply.The proteins from barley (Duff et al, 1997, J. Biol Chem 272:16746-16752) and rice (Arrendondo-Peter et al, 1997, Plant Physiol115:1259-1266) and AHB1 from Arabidopsis (Trevaskis et al, 1997, ProcNatl Acad Sci 94:12230-12234) have been shown to have high oxygenavidity, with dissociation constants for oxyhemoglobin of 2.86 nM, 0.55nM and 1.6 nM respectively, resulting in conditions whereby the freeprotein will remain oxygenated at oxygen concentrations far below thoseat which anaerobic processes are activated. Thus, while roles for Hb inthe facilitated diffusion and sensing of oxygen have been proposed(Appleby, 1992), it is unlikely that these hemoglobins would function aseither facilitators of oxygen diffusion or sensors of oxygen, unless theoxygen avidity was modified by interaction with another component withinthe cell. Thus, while Hb or Hb related proteins are found in alldivisions of living organisms, their function has not been well defined.

Herein, it is shown that nonsymbiotic hemoglobins function to maintainthe energy status of cells exposed to low oxygen tensions and that thisproperty may be a common feature throughout evolution, either duringexposure to hypoxia or under high energy demand.

SUMMARY OF THE INVENTION

According to one aspect of the invention there is provided a recombinantexpression system capable, when transformed into an organism, ofexpressing a gene encoding a nonsymbiotic hemoglobin, which systemcomprises a nucleotide sequence encoding said nonsymbiotic hemoglobinoperably linked to control sequences effective in said organism.

The control sequences may include a strong constitutive promoter.

The nonsymbiotic hemoglobin may be barley hemoglobin.

The organism may be a plant. The plant may be maize.

Preferably, the promoter is maize ubiquitin promoter.

The organism may be a bacteria. The bacteria may be an obligate aerobe.The obligate aerobe may be P. aeruginosa.

According to a second aspect of the invention, there are provided cellstransformed with any one of the expression systems described above.

According to a third aspect of the invention, there is provided atransgenic organism whose genome has been modified to contain theexpression system described above.

According to a fourth aspect of the invention, there is provided amethod of increasing tolerance to hypoxic conditions comprising:

providing an organism having increased cellular levels of anoxygen-binding protein having a low dissociation constant for oxygen;and

placing the organism under hypoxic conditions,

wherein the oxygen-binding protein acts to maintain cellular energystatus during the hypoxic conditions by making oxygen available forcellular metabolism at low oxygen tension.

According to a fifth aspect of the invention, there is provided a methodof lowering the level of fermentation products in an organismcomprising:

providing an organism having increased cellular levels of anoxygen-binding protein having a low dissociation constant for oxygen;and

reducing the level of fermentation products in the cells of the organismby maintaining cell energy status such that fermentation is bypassed.

According to a sixth aspect of the invention, there is provided a methodof maintaining cellular metabolism under hypoxic conditions comprising:

providing an organism having increased cellular levels of anoxygen-binding protein having a low dissociation constant for oxygen;and

placing the organism under hypoxic conditions,

wherein the oxygen-binding protein acts to maintain cellular metabolismstatus by providing oxygen for cellular metabolism.

According to a seventh aspect of the invention, there is provided amethod of increasing oxygen uptake of an organism comprising:

providing an organism having increased cellular levels of anoxygen-binding protein having a low dissociation constant for oxygen;and

exposing the organism to an oxygen-containing environment,

wherein the increased cellular levels of the oxygen-binding proteinresults in increased oxygen uptake.

According to an eighth aspect of the invention, there is provided amethod of improving the agronomic properties of a plant comprising:

providing a plant having increased cellular levels of an oxygen-bindingprotein having a low dissociation constant for oxygen; and

growing the plant.

The improved agronomic properties may include germination, seedlingvigour, reduced cellular levels of fermentation products, increasedoxygen uptake, and increased tolerance to hypoxic conditions.

According to a ninth aspect of the invention, there is provided a methodof performing skin grafts comprising:

isolating skin cells from a patient;

transfecting the skin cells with an expression system comprising anucleotide sequence encoding an oxygen binding protein having a lowdissociation constant for oxygen operably linked to control sequenceseffective in skin cells;

culturing the skin cells such that the oxygen binding protein isexpressed; and

grafting the skin cells onto a region of skin tissue attached to thepatient.

According to a tenth aspect of the invention, there is provided a methodof transplanting an organ from a donor to a recipient comprising:

providing an organ for transplant;

infusing the organ with an oxygen binding protein having a lowdissociation constant for oxygen, thereby improving oxygen supply to theorgan; and

transplanting the organ into the recipient.

The oxygen binding protein having a low dissociation constant for oxygendescribed in the above methods may be a nonsymbiotic hemoglobin. Thenonsymbiotic hemoglobin may be barley hemoglobin.

According to an eleventh aspect of the invention, there is provided amethod of selecting seeds for breeding to produce seed lines havingdesirable characteristics comprising:

providing a representative seed of a given seed line;

growing the seed such that the seed germinates;

isolating an extract from the seed;

measuring levels of hemoglobin expression within the extract; and

selecting or rejecting the seed for further breeding based on thehemoglobin levels.

According to a twelfth aspect of the invention there is provided amethod of determining if a seed is germinating comprising:

providing a seed suspected of germinating;

isolating an extract from the seed; and

measuring levels of hemoglobin expression within the extract, whereinhigh levels of hemoglobin expression indicate that the seed isgerminating.

One embodiment of the invention will now be described in conjunctionwith the accompanying figures in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram summarizing the structures of pAS1 andpAS2 respectively.

FIG. 2 is the protein immunoblot analysis of hemoglobin expression inwild-type (BMS), HB⁺ and HB⁻ maize cell lines with recombinant barleyhemoglobin-specific antibody.

FIG. 3 is a graph of the growth rate of wild-type (BMS), HB⁺ and HBmaize cell lines under normal atmospheric conditions.

FIG. 4 is a bar graph comparison of oxygen uptake by maize wild-type(BMS), HB⁺ and HB⁻ cells.

FIG. 5 is a bar graph comparison of ATP levels in wild-type (BMS), HB⁺and HB⁻ maize cells grown under normal atmospheric conditions, after 12hours of treatment with nitrogen, under normal atmospheric conditionsfollowing treatment with Antimycin A and after 12 hours of treatmentwith nitrogen following treatment with Antimycin A.

FIG. 6 is a bar graph comparison of CO₂ evolution by maize cellscultured under a nitrogen atmosphere.

FIG. 7 is a graph of alcohol dehydrogenase activity in maize cellscultured under a nitrogen atmosphere.

FIG. 8 is a bar graph of oxygen uptake by maize cells under low oxygenatmosphere.

FIG. 9 is a bar graph of oxygen uptake by maize cells under normal airconditions.

FIG. 10 is a graph of cell culture growth following hypoxic treatment.

FIG. 11 is a bar graph of the amount of hemoglobin in crude extractsmade from germinating barley seeds.

FIG. 12 is a Western blot of proteins from transformed and wild type P.aeruginosa. Each lane consisted of 80 μg of crude protein extract fromP. aeruginosa cells and the blot was probed with affinity purifiedbarley Hb antibodies. Lane 1 contains protein extracted from bacteriatransformed with the Hb expression vector, whereas Lane 2 containsprotein extracted from wild-type bacteria.

FIG. 13 is a Northern blot of RNA extracted over time from a germinatingseedling.

Table 1 is a summary of measurements of energy charge and totaladenylates in maize cells before and after exposure to a nitrogenatmosphere for 12 hours.

Table 2 is a summary. of A₆₀₀ measurements of transformed anduntransformed E. coli and P. aeruginosa cells grown aerobically oranaerobically. Measurements are the averages of two separatedeterminations which did not vary by more than 15%.

Table 3 is a summary of ATP measurements of transformed anduntransformed E. coli and P. aeruginosa cells grown aerobically andanaerobically. Measurements are the results of duplicate. assays fromthree separate experiments. Standard error in all cases was no greaterthan 10%.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference.

Expression plasmids containing DNA encoding a nonsymbiotic hemoglobinwere constructed. These plasmids also included a strong constitutivepromoter and a selectable marker compatible with the specific hostorganisms such that when these plasmid constructs were transformed intothe host organisms, the constructs expressed elevated levels of Hbprotein compared to wild type cells. In all cases, the transformed cellshad an elevated level of ATP. This strongly suggests that nonsymbiotichemoglobin functions in maintaining ATP levels and is involved inprimary energy metabolism. Thus, cells engineered to express a higherlevel of Hb will survive longer under low oxygen tension or high energydemand. In other words, the cells maintain vigour and hardiness understressful conditions and can better adapt to varying growth conditions.That is, transformed crop plants containing elevated levels of thenonsymbiotic hemoglobin gene may exhibit increased crop yields due tothe ability of the plant to more effectively survive periods offlooding, the ability of the seed and seedling to develop morevigorously under adverse germination and/or growth conditions, and theability of winter crops to survive ice cover more effectively.Furthermore, given that the effect of nonsymbiotic hemoglobin on cellenergy status is seen in both bacteria and plants, it seems likely thatthis phenomenon is universal. This would in turn mean that nonsymbiotichemoglobins have potential applications in a number of medicalprocedures. For example, skin cells from burn victims are frequentlycultured for transplantation back to the burn victim. Given that oxygensupply is a limiting factor for growth and survival of the transplantedskin grafts, skin cells transfected with nonsymbiotic hemoglobin maypossess improved growth and survival. Similarly, oxygen supply is also alimiting factor in other medical procedures, for example, organtransplants. That is, it is likely that organs possessing nonsymbiotichemoglobins may have enhanced survival following transplant.Furthermore, the hemoglobin gene itself is shown to be expressed at timeof germination, meaning that the hemoglobin gene may be used as a markerfor germination and also as a marker for breeding. That is, levels ofhemoglobin in specific seed lines may be used to select seeds fordeveloping progeny seeds capable of expressing either higher or lowerlevels of hemoglobin.

In one embodiment, expression plasmids containing DNA encoding barleyhemoglobin in both the sense and anti-sense orientation wereconstructed. The plasmids also included the maize ubiquitin promoter,and a selectable marker for selection of transformants, in thisembodiment, a herbicide resistance gene (Bar), conferring resistance toglufosinate ammonium. The plasmids were transformed into cultured maizecells of the Black Mexican Sweet (BMS) variety, producing a cell linecontaining the sense plasmid (HB⁺) and a cell line containing theantisense plasmid (HB⁻).

When grown in an air environment, the HB⁺ and HB⁻ cells did not differsignificantly from wild-type BMS cells in terms of growth rate, oxygenconsumption or cellular ATP levels. However, when grown under a nitrogenatmosphere, ATP levels in the HB⁺ cells remained essentially the same asthose. observed under normal atmosphere conditions while ATP levelsdropped significantly in wild-type and HB⁻ cells. Analysis of ATP levelsin all three cell lines under a nitrogen atmosphere following treatmentwith Antimycin A (which blocks mitochondrial electron transport)indicated that the increase in ATP in HB⁺ cells was notcytochrome-mediated. Furthermore, measurements of CO₂ evolution andalcohol dehydrogenase activity in HB⁺ cells suggested lower ethanolicfermentation rates in this cell line.

These data indicate that over-expression of nonsymbiotic hemoglobinshelps maintain the energy status of cells grown at low oxygen tensions.This in turn has several possible applications, as cells capable ofmaintaining energy status at low oxygen tensions would have, forexample, increased tolerance to a low oxygen atmosphere, improvedgermination rates and seedling vigour, increased ability to maintaincellular metabolism at low oxygen tension, reduced levels offermentation products within the cells due to lowered alcoholdehydrogenase activity, increased oxygen uptake under low oxygen tensionand increased tolerance to hypoxic conditions such as, for example, iceencasement, flood and growth in compacted soil.

EXAMPLE I Plant Cell Cultures

Black Mexican Sweet (BMS) (wild-type), HB⁺ and HB⁻ maize cells werecultured in 250 ml flasks as cell suspensions in 50 ml of MS medium(Murashige and Skooge, 1962, Physiol Plant 15:473-497) macro and microelements supplemented with thiamine 0.5 mg/liter, L-asparagine 150mg/liter, 2,4-dichlorophenoxyacetic acid 2 mg/liter and sucrose 20g/liter. Cultures were shaken at 150 rpm at 25° C. Cells weresubcultured every 7 days. Nitrogen treatment was applied by replacingair in culture flasks with nitrogen and closing the flasks with rubberstoppers, otherwise culture flasks were closed with caps allowing forfree exchange of air. Antimycin A was added as a 27 mM stock solution in2-propanol to give a final concentration of 0.2 mM. Cell samples werecollected by filtration. Cell samples used for adenylate measurementswere immediately frozen in liquid nitrogen and stored at −80° C. untilused.

EXAMPLE II Construction of Plant Expression Vectors

SalI/NotI digested and end-filled barley hemoglobin cDNA was cloned intoBamHI digested and end-filled pAHC17 plasmid (Christensen and Quail,1996, Transgenic Research 5:213-218) in sense and antisense orientationto generate pAS1 (sense) and pAS2 (antisense) plasmids. An EcoRIdigested, end-filled with synthetic HindIII linker, 1.35 kb 35S promoter—bar gene— 35S terminator fragment from pDB1 (Becker et al, 1994, PlantJ 5:299-307) was inserted into HindIII digested pAS1 and pAS2, asdescribed below.

EXAMPLE III Plant Cell Transformation and Selection

A silicon carbide fibres-mediated transformation system was used asdescribed in Kaeppler et al, 1992, Theor App Genet 84:560-566 totransform BMS maize cells with pAS1 and pAS2 vectors. Resistant colonieswere selected on culture medium solidified with 0.2% Phytagel™ (Sigma)and supplemented with glufosinate ammonium at a concentration of 5mg/liter.

EXAMPLE IV Plant Protein Immunoblots

SDS gel electrophoresis, protein transfer to nitrocellulose membrane andantibody detection were performed according to standard Bio-Rad protocol(Bio-Rad bulletin 1721). Hemoglobin protein in transformed lines wasdetected by immunoblots; using a polyclonal antibody raised againstbarley recombinant hemoglobin. Protein concentration was calculated bydensitometric comparison of immunoblots (in four repetitions) with astandard curve of known concentrations of recombinant hemoglobin using aSharp Diversity 1 PDI-3250E Scanner™.

EXAMPLE V Measurement of Plant Growth Parameters

Culture growth was measured by sedimentation in 25 ml graduatedpipettes. Adenylates were extracted in 1N perchloric acid from frozencell samples at −10° C. and ATP, ADP and AMP assayedspectrophotometrically by established protocols as described in Lowryand Passonneau, 1972, A Flexible System of Enzymatic Analysis, AcademicPress: New York.

Alcohol dehydrogenase activity was measured in the ethanol-acetaldehydedirection in fresh cell extracts. Enzyme extraction andspectrophotometric measurements were performed as described in Hansonand Jacobsen, 1984, Plant Physiol 75:566-572.

For measurements of CO₂ evolution from cell cultures, 1 ml gas sampleswere collected with an air tight syringe, from stoppered culture flasks,and analyzed by gas chromatography (Shimadzu GC-8AlT™).

Oxygen uptake was measured polarographically with an O₂ electrode (RankBrothers, Cambridge, UK) for 5 to 30 minutes. The incubation cellcontained 2 ml of culture medium, 0.2 ml (sedimented cell volume) ofcells. In some measurements, 0.2 mM Antimycin A was added, as describedbelow.

EXAMPLE VI Effect of Nonsymbiotic Hemoglobin on Plant Cell Energy Status

As noted above, cultured maize cells of the Black Mexican Sweet (BMS)variety were transformed with a barley hemoglobin gene to observe theeffect of increasing or decreasing hemoglobin expression on cellmetabolism. Specifically, transformation vectors, shown in FIG. 1, wereprepared containing the open reading frame of a barley hemoglobin cDNAin sense and antisense orientations, which were placed under the controlof a strong constitutive promoter, in this embodiment, the maizeubiquitin (Ubi1) promoter. A herbicide resistance gene (Bar), conferringresistance to glufosinate ammonium, was cloned head to tail with thehemoglobin gene constructs to enable selection of transformed celllines. Twenty-four independently transformed sense (pAS1) andthirty-eight anti-sense (pAS2) lines were obtained. Transformation wasconfirmed by Southern blbt analysis and PCR. A sense line (HB⁺)expressing hemoglobin at levels 10 fold higher than wild type (BMS) andan antisense line (HB⁻) with 10 times lower expression of hemoglobinthan BMS, as shown in FIG. 2, were selected for further studies, asdescribed below.

The three cell lines, grown in an air environment, did not differsignificantly from one another with respect to culture growth rates, asshown in FIG. 3, and consumption of oxygen, as shown in FIG. 4.Furthermore, steady state ATP levels were essentially the same in thethree types of cells, as shown in FIG. 5. However, after incubation ofthe cells for a further 12 hours under an atmosphere of nitrogen gas,significant differences were observed in the ATP levels of the celltypes. Specifically, the level of ATP was highest in HB⁺ cells, beingonly marginally lower than under normal atmospheric conditions while ATPlevels in wild type (BMS) cells were 27% lower than HB⁺ cells and ATPlevels in HB⁻ cells were 61% lower than HB⁺ cells. Differences in energycharge and total adenylates were also observed in cells exposed tonitrogen atmospheres, as summarized in Table 1. As can be seen, energycharge was relatively the same in all three cell types under normalatmospheric conditions and in BMS and HB⁺ cell lines after 12 hours of anitrogen atmosphere. HB⁻ cells, on the other hand, were unable tomaintain energy charge during the 12 hour exposure to a nitrogenatmosphere. Total adenylates remained the same in all three cell linesunder atmospheric conditions and in HB⁺ cells in a nitrogen atmosphere;however, in BMS and HB⁻ cells, the total adenylates declined by about 35percent.

From this, it is evident that determining what part of the cell'smetabolism contributes to this increased ability to maintain energystatus in the presence of hemoglobin is critical to understanding therole of nonsymbiotic hemoglobin. To examine the possibility thathemoglobin might provide oxygen to generate ATP via cytochrome-mediatedrespiratory processes, Antimycin A (0.2 mM), which blocks mitochondrialelectron transport in the span from cytochrome b to c and has been shownto induce hemoglobin expression in aleurone layers (Nie and Hill, 1997,Plant Physiol 114:835-840) was used. Antimycin A inhibited 80% of theoxygen uptake by maize cells within 30 minutes of treatment. After 12hours exposure to Antimycin A in an air environment, ATP levels in thethree cell types were similar to those of untreated cells after 12 hoursunder a nitrogen atmosphere, as shown in FIG. 5. However, upon placingAntimycin A-treated cells in a nitrogen atmosphere for 12 hours, thecell lines all showed decreases in ATP but, consistent with the previousexperiments, the levels of ATP decreased in the order HB⁺, BMS, and HB⁻.This provides evidence that the increase in ATP brought about by thepresence of hemoglobin was not the result of cytochrome-mediatedmitochondrial respiration. It is also unlikely that the increased ATP isthe result of oxyhemoglobin supporting mitochondrial alternative oxidaseactivity, which would increase substrate phosphorylation throughglycolysis.

Furthermore, as shown in FIG. 6, CO₂ evolution from hypoxic HB⁺ cellswas 20 to 30% lower than CO₂ levels evolved from BMS or HB⁻ cells, whichwould not be anticipated if the Krebs cycle was being maintained throughalternative oxidase activity.

EXAMPLE VII Plant Cell Alcohol Dehydrogenase Levels

An examination of alcohol dehydrogenase activity (ADH) in the cell linesshowed that ADH increased in all three lines over the course of theexperiments, but the ADH activity was significantly lower in the sensetransformants (HB⁺) than in antisense transformants (HB⁻) or wild-typecells, as shown in FIG. 7. Fluorescein diacetate staining(Heslop-Harrison et al, 1984, Theor Appl Genet 67:367-375) showed nodifference in the viability of the cell lines at the end of theincubation period. The reduced ADH activity, along with lower CO₂evolution in HB⁺ cells, likely reflects lower ethanolic fermentationrates, suggesting that a fermentative pathway may be the main source ofcarbon dioxide production in this system.

EXAMPLE VIII Oxygen Uptake by Plant Cells

As discussed above, the presence of nonsymbiotic hemoglobin clearlyaffects the energy status of maize cells under hypoxia. Furthermore,differences between the HB⁺, wild type and HB⁻ cells were observed onlyunder the conditions of limited oxygen. To investigate the possibilitythat the observed differences may be due to the different abilities ofthe cell lines to utilize oxygen that is available in lowconcentrations, the oxygen uptake by the maize cells was measured undernormal air conditions, shown in FIG. 9, and in medium equilibrated witha mixture of 2% O₂ and 98% N₂, shown in FIG. 8. Specifically, oxygenuptake was measured polarographically with an O₂ electrode. As can beseen, HB⁺ cells were more efficient at oxygen uptake than the wild-typecells and much more efficient than the HB⁻ cells. Specifically, theoxygen uptake by the HB⁺ cells from the medium equilibrated with 2%oxygen was 55% of that of all three cell lines under normal airconditions, as shown in FIGS. 8 and 9. Furthermore, wild-type BMS andHB⁻ cells grown at 2% O₂ exhibited O₂ uptake at 44% and 18% respectivelyof the oxygen uptake of the cell lines grown under normal conditions, asshown in FIGS. 8 and 9. These results clearly indicate that the rate ofoxygen utilization by maize cells under low oxygen atmosphere depends onthe presence of the non-symbiotic hemoglobin.

EXAMPLE IX Plant Cell Growth After Exposure to Hypoxic Stress

The ability of the cell cultures to continue growth after exposure tohypoxic stress was also tested. Maize cell cultures were placed underthe atmosphere. of nitrogen for 12 and 24 hours, then cells wereharvested, transferred to a fresh medium and their growth was monitoredby sedimented cell volume measurements, as shown in FIG. 10. Uponplacement under the N₂ atmosphere, the cell growth of all three celllines ceased, but resumed after transfer to the fresh medium and normalatmospheric conditions. However, while the HB⁺ cell cultures resumedgrowth almost immediately after the transfer to normal air conditions,the HB⁻ cells showed a 36 hour lag period before commencement ofintensive growth. Furthermore, the growth of the wild-type cultures,during the first 36 hours after the transfer to normal conditions, wasslower than that of HB⁺ cells, as shown in FIG. 10. It is of note thatafter the initial 36 hour period, the growth rates of the three celllines were almost identical. The differences in cell volume at each timepoint were most likely a result of the growth activity during thisinitial period. The culture re-growth after the 24 hour hypoxic exposurewas the same for all three cell lines, as after the 12 hour treatment.The observed differences may be explained by different levels of cellsurvival under stress, and, depending on the cell line, the same cellvolume could contain different numbers of growing cells. On the otherhand, the increased growth rates of the HB³¹ and the wild-type BMScultures after a lag period, shown in FIG. 10, suggests a longer stressrecovery period rather than cell death.

EXAMPLE X Hemoglobin Expression in Germinating Barley

Polyclonal antibodies to purified recombinant barley hemoglobin wereraised in rabbits and used to. investigate the expression of hemoglobinin monocotyledonous plants. Specifically, hemoglobin was shown to beexpressed in whole seeds, as shown in FIG. 11, embryo-less half seedsand excised embryos during germination. The fact that hemoglobin wasexpressed in both embryo-less half seeds and excised embryos indicatesthat the gene is independently responsive to signals in both tissues andsuggests that both the aleurone layer and the embryo may experienceoxygen deficiencies during the imbibition process. In the excisedembryo, hemoglobin was induced between 4 and 6. hours after imbibition.Since germination and the early stages of seedling growth are known tobe periods of high metabolic demand (Bewley and Black, 1990, ProgNucleic Acid Res Mol Biol, 38:165-193, incorporated herein byreference), this data is consistent with the proposed. concept that ademand on energy charge or ATP requirement is primarily responsible forhemoglobin induction (Nie and Hill, 1997, Plant Physiol 114:835-840).Major changes in ATP content of the embryos did occur within one hourafter imbibition, which is consistent with previous reports. Proteinhydration, protein synthesis. and nucleotide synthesis are among thefirst events of germination. These early -events, which consume largeamounts of ATP, may well be a factor in the observed induction ofhemoglobin synthesis at 4 to 6 hours after imbibition. However,induction occurs well before the major increase in α-amylase secretion,a period of high metabolic demand, and so the relationship betweenhemoglobin synthesis and energy availability needs furtherclarification.

In half seeds, there is an apparent induction of hemoglobin duringimbibition, without the use of gibberellic acid to stimulate thesynthesis of hydrolytic enzymes. Furthermore, isolated aleurone layersdo not .show appreciable amounts of hemoglobin unless induced by anoxiausing a nitrogen environment (Nie and Hill, 1997). The aleurones inthese half-seeds may well be experiencing anoxia due to entrapment inthe endosperm and seed coat.

Thus, to summarize, very little or no hemoglobin expression was observedin dry barley seeds but germination resulted in the expression ofhemoglobin which peaked at 2-3 days after imbibition, as shown in FIG.11. Furthermore, hemoglobin expression was also observed in maize,wheat, wild oat and Echinochloa crus galli seeds during germination.Dissection of tissues from the barley seedlings showed that most of thehemoglobin was expressed in the root and seed coat (aleurone layer),with very little in the coleoptile. Imbibition of half seeds or excisedembryos resulted in the expression of hemoglobin. ATP measurements ofbarley embryos showed that ATP levels quickly increased afterimbibition. α-Amylase activity was also determined in the embryos tocorrelate hemoglobin expression with a well-characterized germinationresponse. The results demonstrate that hemoglobin expression is a normalconsequence of germination.

In addition, whole barley seeds were imbibed for 16 hours at 22° C.Embryos were excised from the caryopis after 2, 4, 8, 10, 12, 14 and 16hours imbibition. It was noted that radicle protrusion occurs after 8hours. The embryos were ground in liquid nitrogen and RNA extracted forNorthern analysis using an RNA probe transcribed from barley Hb cDNA. Ascan be seen in FIG. 13, it was found that no message was present inunimbibed seeds but was detectable after just two hours imbibition.Expression increased up until 8 hours when radicle emergence occurred.The amounts of message then decreased for the next 8 hours. Theseexperiments show that hemoglobin expression occurs during germination.As such, it is clear that hemoglobin expression can be used as a markerfor germination.

EXAMPLE XI Construction of Bacterial Expression Constructs

A recombinant Hb cDNA-containing pUC19 construct (Duff et al, 1997) wasused as the starting material. The Hb cDNA was excised from the pUC19construct by digestion with the restriction enzymes EcoRI and HindIII.The insert was then ligated into the pPZ375 multiple cloning sitebetween HindIII and EcoRI such that the coding sequence was in thecorrect reading frame.

EXAMPLE XII Transformation and Screening of Recombinant E. coli

Escherichia coli DH5α cells were then transformed with the pPZ375-Hbconstruct according to the instructions for the Canadian LifeTechnologies subcloning efficiency competent cells, incorporated hereinby reference. It is of note that in this instance Blue-White screeningwas unnecessary. E. coli cells were plated, screened and grown aspreviously described (Duff et al, 1997). Plasmid DNA was prepared fromthe cells using the small scale preparation protocol (Sambrook et al,1989). The recombinant plasmid was then used to transform competentPseudomonas aeruginosa, as described below.

EXAMPLE XIII Preparation and Transformation of Competent Pseudomonasaeruginosa

100 ml of LB media in a 500 ml flask was inoculated with 1 ml of anovernight culture of Pseudomonas aeruginosa and grown for 2.5 hours to acell density of approximately 108 cells/ml. Cells were harvested bycentrifugation at 1000 g and then resuspended in 10 ml of CompetencyBuffer (0.15 M MgCl₂, 15% (v/v) glycerol; 1Q mM Pipes (Sigma), pH 7.0).Cells were incubated in an ice water bath for 5 minutes, pelleted at1000 g, and resuspended in 10 ml of Competency Buffer. Cells were thenincubated in an ice water bath for 20 minutes, pelleted at 1000 g, andresuspended in 10 ml of Competency Buffer. Cells were then frozen at−70° C. until used for transformation. DNA (approximately 0.2 μg of therecombinant plasmid) was used to transform 200 μl of competentPseudomonas aeruginosa cells. Cells were incubated in an ice water bathfor 60. minutes and heat shocked for 3 minutes at 37° C. while gentlyrocking the tube. Cells were placed in an ice water bath for 5 minutes.0.5 ml of room temperature LB broth was added and the cells wereincubated at 37° C. for 2.5 hours with no rotation. Cells wereconcentrated by centrifugation and plated on appropriate media.

EXAMPLE XIV Electrophoresis and Bacterial Protein Immunoblotting

DNA agarose electrophoresis, protein acrylamide electrophoresis andprotein immunoblotting was performed as previously described above.

EXAMPLE XV Bacterial Growth and Treatment

E. coli was inoculated into four 400 ml cultures and grown for 3 hours.After 3 hours, A₆₀₀ was measured as an estimate of bacterial growth andthen either air or nitrogen was bubbled through the media for 5 minutesand the flasks were sealed. The bacteria were grown for a further 6hours after which the A₆₀₀ was determined for each flask as an estimateof bacterial growth. Similarly, P. aeruginosa was inoculated into four400 ml cultures and grown for 3 hours using the same protocol asdescribed above for E. coli,

EXAMPLE XVI ATP Extraction and Assay

ATP was extracted and assayed according to standard procedures known inthe art (Lowry and Passonneau, in A Flexible System of EnzymaticAnalysis (1972, Academic Press: New York) pp 146-222, incorporatedherein by reference).

EXAMPLE XVII Expression of Barley Hb in E. coli and P. aeruginosa

Untransformed E. coli cells and E. coli cells previously transformedwith Hb cDNA were used (Duff et al., 1997). Western blot analysisconfirmed that both E. coli (data not shown) and P. aeruginosa (FIG. 12)had been successfully transformed and were expressing significantamounts of Hb. Recombinant E. coli and P. aeruginosa were also visuallymore red than their wild type counterparts (data not shown). Levels ofrecombinant barley hemoglobin expressed in the two species of bacteriawere roughly equal based on SDS-PAGE and protein immunoblot analysis.

EXAMPLE XVIII

Growth Rates of E. coli and P. aeruginosa

The A₆₀₀ measurements of 400 ml cultures of transformed anduntransformed E. coli and P. aeruginosa grown under both aerobic andanaerobic conditions are shown in Table 2. E. coli containing therecombinant plasmid grew considerably slower than bacteria containingpUC19. There were no differences in growth between bacteria grown underair or anoxic conditions for E. coli containing either plasmid. P.aeruginosa containing the recombinant plasmid also grew somewhat slowerthan the bacteria containing pUC19. However, anoxic treatment virtuallystopped the growth of both the wild type and recombinant obligateaerobic bacteria P. aeruginosa.

EXAMPLE XIX ATP Levels in E. coli and P. aeruginosa

ATP levels from aerobically and anaerobically grown E. coli and P.aeruginosa are shown in Table 3. As can be seen, E. coli cells had thesame total ATP regardless of whether or not they were expressing barleyHb or whether they were grown under aerobic or non-aerobic conditions.However, P. aeruginosa containing the recombinant barley Hb hadsignificantly higher levels of ATP under both aerobic and non-aerobicconditions. These results are not surprising, given that E. coli readilyadapts to grow in environments with limited oxygen. P. aeruginosa, onthe other hand, is an obligate aerobe and is unable to grow inenvironments with limited oxygen. Furthermore, it is known that ATPlevels and energy charge are directly related to the-metabolic state ofan organism and that organisms with low ATP levels and energy charge aregenerally considered to be under stress or in a state of dormancy. Thus,the fact that P. aeruginosa containing nonsymbiotic hemoglobin has animproved energy status is evidence that the presence of this proteinfacilitates adaptation to low oxygen tension.

Discussion

Higher plant hemoglobins are cytoplasmic proteins (Wittenberg andWittenberg, 1990). With this in mind, transformation constructs weredesigned for cytoplasmic expression of hemoglobin. Barley hemoglobincDNA hybridizes to only one locus in barley and maize genomes (Taylor etal, Plant Mol Biol 24:853-862) and, therefore, -sense and antisenseexpression of this cDNA would not be expected to affect the expressionof any other genes. It is of note that the polyclonal anti-hemoglobinantibody used was raised and titrated against recombinant barleyhemoglobin. Furthermore, it is clear that there is over and underexpression of hemoglobin in the transgenic cells.

The lack of effect of hemoglobin on cell growth and oxygen uptake undernormal air-conditions likely reflects the fact that barley (Taylor etal, 1994) and maize hemoglobin genes are induced under conditions oflimited oxygen availability, resulting in the protein having littleeffect when oxygen supplies are not impaired. The results, however, showclearly that the energy status of maize cells when oxygen is limiting isaffected by the ability of the cells to produce hemoglobin. Totaladenylates and ATP levels are maintained during the period of exposureto limiting oxygen when hemoglobin is constitutively expressed in thecells. Alternatively, when hemoglobin expression is suppressed byconstitutive expression of antisense barley hemoglobin message, thecells are unable to maintain their energy status during oxygenlimitation. In wild-type (BMS) cells, it would appear that the inductionof native maize hemoglobin was sufficient to maintain the energy charge,but not the total adenylate pool. This is consistent with theobservation that a decline in the adenylate pool has been noted duringhypoxia in maize root tips (Saint-Ges et al, 1991, Eur J Biochem200:477-482). Under limiting oxygen, plant cells turn their metabolismtowards fermentation in order to oxidize NADH necessary to maintainglycolytic substrate phosphorylation. Lower alcohol dehydrogenaseactivity in HB⁺ cells suggests that hemoglobin provides an alternativeto potentially harmful fermentation. Specifically, carbon dioxide isproduced by the HB⁺ cells in lower amounts than by HB⁻ and wild-typemaize cells, reflecting lower ADH activity and suggesting that theethanolic fermentation is the only source of CO₂. The dissociationconstant of barley oxyhemoglobin is about 3 nM (Duff et al, 1997),indicating that oxyhemoglobin, acting alone, would be ineffective inproviding oxygen to maintain mitochondrial respiratory processes. Thisis confirmed by the observation that Antimycin A has no effect on theability of hemoglobin-containing cells in maintaining their energystatus under low oxygen tensions. The results discussed above suggestthat hemoglobin maintains energy status of. the cell by means differentfrom mitochondrial oxidative phosphorylation , probably by facilitatingglycolysis to generate ATP through substrate level phosphorylation.

It is of note that hemoglobins of barley (Taylor et al, 1994) and maizeas well as Arabidopsis AHB1 (Trevaskis et al, 1997) are hypoxiainducible. Furthermore, it has been demonstrated that, in barleyhemoglobin this is not due to a lack of oxygen per se, but in responseto insufficient mitochondrial ATP synthesis. In addition, nonsymbiotichemoglobins are expressed in metabolically active tissues such as roots(Taylor et al, 1994; Arredondo-Peter et al, 1997; Trevaskis, 1997),aleurone (Taylor et al, 1994), vascular tissues of leaves, stems andseedling cotyledons (Andersson et al, 1996, Proc Natl Acad Sci93:5682-5687). Taken together, these data support a hypothesis thatnonsymbiotic hemoglobins utilize available oxygen to maintain the cell'senergy status in cells exposed to low oxygen tensions or otherconditions that reduce cellular ATP levels. The very low dissociationconstant of barley oxyhemoglobin makes it an ideal candidate forsequestering oxygen in low oxygen environments. Interaction with anothercompound, perhaps a flavoprotein, could create a complex capable ofoxidizing NADH, in a manner analogous to Hmp protein of E. coli (Pooleet al, 1996, Microbiology (Reading) 142:1141-1148). This would providean efficient means of oxidatively regenerating NAD to supportglycolysis, bypassing the fermentative route to ethanol.

The effects of expression. of sense and antisense hemoglobin on energycharge are reminiscent of hypoxic acclimation of plant tissues, forexample, maize root tips, which develop a tolerance to short term anoxiaif they have been acclimated by exposure to hypoxic conditions (Johnsonet al, 1969, Plant Physiol 91:837-841). Specifically, acclimation isaccompanied by increased energy charge (Hole et al, 1992, Plant Physiol99:213-218) resulting from a sustained glycolytic rate compared tonon-acclimated root tips (Xia and Saglio, 1992, Plant Physiol 100:40-46;Xia and Roberts, 1996, Plant Physiol 111:227-233). Similarly, wintercereals show increased survival to hypoxia caused by ice encasement ifthey have been acclimated by exposure to hypoxic conditions (Andrews andPomeroy, 1983, Can J Bot 61:142-147). Acclimated plants maintain higherlevels of adenylates and ATP during ice encasement, as a result ofaccelerated rates of glycolysis, than non-acclimated plants (Andrews andPomeroy, 1989, Plant Physiol 91:1063-1068). Maximum induction of barleyhemoglobin message occurs within 12 hours exposure to hypoxic conditions(Taylor et al, 1994), which is well within the time interval used foracclimation in the above examples. Furthermore, it has been shown thatthe expression of hemoglobin is not directly influenced by oxygen usageor availability but it is influenced by the availability of ATP in thetissue (Nie and Hill, 1997). This suggests that the increased survivalof plants to anoxia as a result of hypoxic acclimation is a consequenceof hemoglobin gene expression induced by declining ATP levels duringacclimation.

From an evolutionary standpoint, it has been suggested that nonsymbiotichemoglobins represent one. of the more ancient forms of planthemoglobins (Andersson et al, 1996). Evidence presented here addscredence to this idea. Since early life on earth existed in oxygen-poorenvironments, the presence of a hemoglobin capable of utilizing oxygen.at low oxygen tensions would have provided an evolutionary advantage toan organism. Oxygen produced during photosynthesis and retained asoxyhemoglobin would provide a source of oxygen to oxidize NADH,maintaining a high glycolytic flux during darkness to provide ATP forcell growth and development.

The high oxygen avidity of hemoglobin (Arredondo-Peter et al, 1997; Duffet al, 1997; Trevaskis et al, 1997) argues against hemoglobinfunctioning to facilitate diffusion of oxygen. Because the hemoglobinwill be induced intracellularly in a highly reductive environment withlow energy charge it is possible that hemoglobin functions as anelectron transport protein similar to cytochrome c. Further work is nowbeing carried out to more closely examine the potential effect of oxygenlimitation and hemoglobin expression during germination.

The function of this enigmatic protein is still far from certain. Wehave observed hemoglobin gene expression (or increases in hemoglobinexpression) unequivocally in at least 4 cases: (1) in intact whole seedsduring germination; (2) in excised embryos and embryo-less half seedsimbibed in water; (3) in aleurone layers which have been stressed by alow oxygen environment or respiratory inhibitors (Nie and Hill, 1997);and (4) in barley roots after flooding (Taylor et al, 1994). In everysituation, it is likely that the ATP requirement of the cell exceeds theATP supply either because of low oxygen supply (such as is the case ofthe flooded plants or stressed seed tissue) or due to high metabolicrates (such as likely to be the case during germination). Hemoglobinexpression seems to be both a normal event during seed germination aswell as an adaptation of plants to low oxygen environments.

As. discussed above, the results obtained from expression of Hb inbacterial cells are reminiscent of maize suspension cells where it washypothesized that Hb might be involved in maintaining the level of ATPthrough the involvement of a pathway other than oxidativephosphorylation. It seems reasonable to conclude that given thesimilarity of results that a similar mechanism might be occurring in P.aeruginosa but not E. coli. As discussed above, this is likely due tothe fact that E. coli adapts readily to grow under conditions of limitedoxygen, whereas P. aeruginosa is an obligate aerobe and does notnormally grow under conditions of limited oxygen. However, the fact thatthis phenomenon is seen in organisms as diverse as plants and aerobicbacteria further suggests that whatever the function of the nonsymbioticplant hemoglobin is, it may be widely represented in nature and may haveevolved from a very ancient and fundamental form of oxidative metabolismwhich evolved before mitochondrial oxidative phosphorylation. This finalconclusion is suggested by the fact that Hb can bind oxygen at levelsfar lower than most other oxygen binding proteins (especially cytochromeC and the alternative oxidase) and may have evolved when oxygen levelsin the atmosphere were much lower.

As will be apparent to one knowledgeable in the art, for expressing Hbin a variety of host organisms, expression vectors may be constructedcontaining Hb linked to a host-specific promoter. Furthermore, theexpression vector may contain a selectable marker functional in thespecific host for selecting transformants. In this manner, a variety ofexpression vectors may be constructed for use in a variety of hostorganisms. Transgenic or recombinant organisms containing these vectorswill have increased tolerance to hypoxic conditions, lower levels offermentation products and increased oxygen uptake. More specifically,plants containing the Hb expression vector described above engineeredfor expression in a given plant will have improved agronomic properties,such as, for example, germination, seedling vigour, reduced cellularlevels of fermentation products, increased oxygen uptake, and increasedtolerance to hypoxic conditions.

Furthermore, given that the effect of nonsymbiotic hemoglobin on cellenergy status is seen in both bacteria and plants, it seems likely thatthis phenomenon is universal. This would in turn mean that nonsymbiotichemoglobins have potential applications in a number of medicalprocedures. For example, skin cells from burn victims are frequentlycultured for transplantation back to the burn victim. Given that oxygensupply is a limiting factor for growth and survival of the transplantedskin grafts, skin cells transfected with nonsymbiotic hemoglobin maypossess improved growth and survival. Similarly, oxygen supply is also alimiting factor in other medical procedures, for example, organtransplants. That is, it is likely that organs possessing nonsymbiotichemoglobins may have enhanced survival following transplant.

As is apparent to one knowledgeable in the art, other oxygen bindingproteins displaying a low dissociation constant for oxygen may be usedin place of Hb in the above-described expression vectors.

Furthermore, as discussed above, the expression of hemoglobin occursduring seedling germination. As such, expression of hemoglobin can beused as a marker for germination. In addition, as discussed above,hemoglobin expression is clearly related to seedling vigour. As such,levels of hemoglobin expression at the time of germination can be usedfor selecting seeds for breeding.

Since various modifications can be made in our invention as herein abovedescribed, and many apparently widely different embodiments of same madewithin the spirit and scope of the claims without department from suchspirit and scope, it is intended that all matter contained in theaccompanying specification shall be interpreted as illustrative only andnot in a limiting sense.

TABLE 1 Energy charge and total adenylates in maize cells before andafter exposure to a nitrogen atmosphere for 12 hours. Results areexpressed as nmol per g fresh weight. Maximum SE (n = 3) was 5%. Totaladenylates Energy Charge (nmol per g fresh weight) Cell Line AirNitrogen Air Nitrogen HB⁺ 0.93 0.93 96 92 Wild 0.94 0.93 94 61 HB⁻ 0.910.73 99 59

TABLE 2 A₆₀₀ measurements of transformed and untransformed E. coli andP. aeruginosa cells grown aerobically and anaerobically. Measurementsare the averages of two separate determinations which did not vary bymore than 15%. E. coli P. aeruginosa Wild type +Hb Wild type +Hb 3 hr O₂0.044 0.040 0.098 0.059 9 hr O₂ 0.147 0.110 1.392 1.074 3 hr O₂ + 6 hrN₂ 0.144 0.102 0.141 0.074

TABLE 3 ATP measurements of transformed and untransformed E. coli and P.aeruginosa cells grown aerobically and anaerobically. Measurements arethe results of duplicate assays from 3 separate experiments. Standarderror was in all cases no greater than 10%. E. coli P. aeruginosa Wildtype +Hb Wild type +Hb 9 hr O₂ 0.019 0.019 0.019 0.025 3 hr O₂ + 6 hr N₂0.018 0.019 0.011 0.018

1. A method of increasing a plant's tolerance to hypoxic conditions,comprising transforming a plant with an expression system comprising anucleic acid molecule encoding a plant nonsymbiotic hemoglobin, whereinthe plant exhibits increased tolerance to hypoxic conditions as comparedto a plant that has not been transformed with a nucleic acid moleculeencoding a plant nonsymbiotic hemoglobin.
 2. The method according toclaim 1, wherein the plant nonsymbiotic hemoglobin is barleynonsymbiotic hemoglobin.
 3. The method according to claim 1, wherein theplant exhibits improved germination under hypoxic conditions, ascompared to a plant that has not been transformed with a nucleic acidmolecule encoding a plant nonsymbiotic hemoglobin.
 4. The methodaccording to claim 1, wherein the plant exhibits improved seedlingvigour under hypoxic conditions, as compared to a plant that has notbeen transformed with a nucleic acid molecule encoding a plantnonsymbiotic hemoglobin.
 5. The method according to claim 1, wherein theplant exhibits reduced cellular levels of ethanolic fermentationproducts under hypoxic conditions, as compared to a plant that has notbeen transformed with a nucleic acid molecule encoding a plantnonsymbiotic hemoglobin.
 6. The method according to claim 1, wherein theplant exhibits increased oxygen uptake under hypoxic conditions, ascompared to a plant that has not been transformed with a nucleic acidmolecule encoding a plant nonsymbiotic hemoglobin.
 7. The methodaccording to claim 1, wherein the hypoxic conditions are related to oneor more conditions selected from the group consisting of ice encasement,flood, and impacted soil.
 8. The method according to claim 1, whereinthe plant exhibits increased ability to maintain total adenylates underhypoxic conditions, as compared to a plant that has not been transformedwith a nucleic acid molecule encoding a plant nonsymbiotic hemoglobin.9. The method of claim 1, wherein the expression system furthercomprises a control sequence operably linked to said nucleic acidmolecule.
 10. The method of claim 9, wherein the control sequence is astrong constitutive promoter.
 11. The method of claim 9, wherein thecontrol sequence is a host-specific promoter.
 12. The method of claim 1,wherein the plant nonsymbiotic hemoglobin is a rice nonsymbiotichemoglobin.
 13. The method of claim 1, wherein the plant nonsymbiotichemoglobin is an Arabidopsis nonsymbiotic hemoglobin.
 14. The method ofclaim 1, wherein the plant nonsymbiotic hemoglobin is a maizenonsymbiotic hemoglobin.
 15. The method of claim 1, wherein the plant isa maize plant.
 16. The method of claim 15, wherein the expression systemfurther comprises a maize ubiquitin promoter.
 17. The method of claim 1,wherein the expression system further comprises a selectable marker. 18.A plant having increased tolerance to hypoxic conditions transformed inaccordance with the method of claim
 1. 19. The plant of claim 18,wherein the plant expresses plant nonsymbiotic hemoglobin at an elevatedlevel under hypoxic conditions as compared to a plant that has not beentransformed with an expression system comprising a nucleic acid moleculeexpressing a plant nonsymbiotic hemoglobin.
 20. The plant of claim 19,wherein the plant expresses plant nonsymbiotic hemoglobin under hypoxicconditions at a level ten times higher than that of a plant that has notbeen transformed with an expression system comprising a nucleic acidmolecule expressing a plant nonsymbiotic hemoglobin.